The ability to manipulate the sex determination pathways during embryonic development so as to favor one sex or the other can have significant economic benefits for the poultry industry. For layer breeders who focus on hens for egg production, the conversion from males to females is of particular concern. Typically, new-born chicks are screened by hand and up to 98% of the males are destroyed. The remaining 2% of males are retained for breeding purposes. Wastage of new-borns and the labor costs of saving the birds are, therefore, a major financial burden. Conversely, broiler production requires predominantly male birds in the flocks because of the 10-15% body weight advantage of male birds compared to female birds.
There is a significant economic incentive, therefore, to identify genes that regulate germ-line development and thereby be used to direct the gender distribution of birds. For example, altering the level of suppression of such a gene in a transgenic animal could drive an embryo down the male or female path.
The gene Mago nashi (grandchildless) was first identified in the fruit fly Drosophila melanogaster, and shown to be involved in the localization and development of germ cells. See Boswell et al., Development 113, 373-384 (1991). In Drosophila, the Mago gene product is necessary for anterior-posterior polarization in the oocyte. (Newmark et al., Development 124, 3197-3207 (1997)). The Mago nashi gene is also required for the continued viability of the Drosophila embryo and the dorsal-ventral migration of the oocyte nucleus to the anteriodorsal position (Newmark et al., Development 124, 3197-3207 (1997)).
Highly conserved homologs of the Drosophila Mago nashi gene are widespread throughout the animal kingdom and in plants, but are not found in fungi or bacteria (Micklem et al., Current Biol. 7, 468-478 (1997)). In animals, Mago-related amino acid sequences have been isolated from Caenorhabditis elegans, Xenopus laevis, Mus musculus (mouse) (Newmark et al., Development 124, 3197-3207 (1997)) and human (Zhao et al., Genomics 47, 319-322 (1998)). In Drosophila, the protein product of Mago nashi is a 147 amino acid (aa) protein. The human homolog MAGOH is a 146-aa protein, the sequence of which has 90% identity to the Drosophila equivalent. While the mouse MAGOH amino acid sequence is the same as the human MAGOH sequence, the nucleotide sequence is only 88% identical (Zhao et al., Genomics 47, 319-322 (1998)). The mammalian MAGOH and C. elegans Mago nashi homolog are only 77% similar in amino acid sequence.
Although the highly conserved mammalian MAGOH proteins display sequence variation when compared to the invertebrate homologs, two regions of the protein are conserved and identical in all species examined. An N-region conserved domain is also found in a related (but partial) amino acid sequence obtained from rice (Oryza sativa) (Zhao et al., Genomics 47, 319-322 (1998)). The overall conservation of the protein, and absolute preservation of the sequence in two domains probably reflects that the Mago protein has central functions key to the survivability of multicellular organisims.
While the activity of the D. melangogaster Mago nashi gene product has been shown as necessary for germ-line development, in humans the MAGOH gene is expressed in all tissues, both adult and fetal, that have proliferating cells. MAGOH is serum inducible in quiescent cells supporting its role in cell proliferation and interacts with an RNA-binding protein (Zhao et al., Genomics 63, 145-148 (2000)).
The role of the MAGOH gene or its homologs in cell development is ill-defined. In Drosophila, at least, it is involved in oogenesis. In humans, it is at high levels in proliferating cells. In C. elegans, however, the mag-1 gene, the homolog of Mago nashi, regulates masculanization of the hermaphrodite germ-line (Li et al, Develop. Biol. 218, 172-182 (2000)). Thus, RNA-mediated interference of mag-1 caused masculanization of C. elegans hermaphrodite, and it has been suggested that mag-1 functions by inhibiting one or more genes participating in mascularization.
The part played by the C. elegans mag-1 gene in determining the sexual development of the worm, and the highly conserved nature of the protein, signifies the importance of identifying the Mago nashi gene, and the expressed product thereof, from the chicken. The identification and isolation of the chicken Mago nashi gene will enable detection of the gene products, including RNA transcripts and protein products, in the various tissues of chicken. Furthermore, isolated Mago nashi-related nucleic acids will enable the manipulation of Mago nashi gene activity so that the gender of a chicken may be selected in favor one sex or the other. Thus, directing the production of either males or females will be to the economic advantage of poultry breeders and allow the successful generation of stable transgenic populations of birds. The similarity of the Mago homologs will enable this technology to be applied to other avian populations yielding economic or conservation benefits.